Huntington's disease-like 2 (HDL2), a devastating neurodegenerative disorder nearly indistinguishable from Huntington's disease (HD), is caused by a CTG/CAG expansion mutation within exon 2A of junctophilin-3 (JPH3). Our preliminary data demonstrates that, like the mutation in myotonic dystrophy 1 (DM1), a disease which involves muscle more than brain, the HDL2 mutation leads to the generation of JPH3 transcripts that contain a long CUG repeat. Also like DM1, the HDL2 mutant transcripts form foci that contain the protein muscleblind1 (MBNL1), a regulator of gene splicing. Our preliminary cell models of HDL2 suggest that JPH3 transcripts with an expanded repeat are neurotoxic. We therefore hypothesize that the neurodegeneration in HDL2 stems from the expression of JPH3 transcripts with a long CUG repeat, and that the pathogenic pathway leading from the repeat expansion to neuronal dysfunction and death involves the sequestration of MBNL1 with subsequent misplicing of a number of downstream genes. We also predict that other CUG repeat binding proteins, including CUGBP1, may influence disease pathogenesis. We will test this hypothesis in a series of experiments in 3 specific aims. In Specific Aim 1, we will develop an inducible stable cell model and a mouse primary neuronal model of HDL2 RNA toxicity. We predict that expression of JPH3 RNA with an expanded repeat, mutated to prevent translation into protein, will result in toxicity and formation of RNA foci in the stable cell model, and will be toxic to cortical and striatal neurons, but not cerebellar neurons. We further predict that the extent of toxicity will depend on the sequence flanking the repeat. In Specific Aim 2, we will examine the pathogenic role of RNA-binding proteins and RNA foci formation in CUG repeat-induced toxicity. We predict that MBNL1 will mediate CUG repeat toxicity, and that neurotoxicity will be influenced by other RNA-binding proteins. In Specific Aims 1 and 2, a parallel set of experiments will compare the pathogenic properties of transcripts with expanded CUG repeats with the pathogenic properties of untranslatable huntingtin transcripts with expanded CAG repeats. In Specific Aim 3, we will develop a bacterial artificial chromosome (BAC) mouse model of HDL2, in which the human JPH3 gene will be expressed under control of its native promoter. We predict that the BAC mice with expanded CTG repeats will develop a neurological and pathological phenotype with similarities to HDL2. We also predict that these mice will develop RNA foci that contain MBNL1, and that the splicing of genes regulated by MBNL1 will be abnormal. Overall, the experiments proposed here will enhance our understanding of HDL2, and potentially HD and DM1. More generally, the experiments will provide new understanding of the little explored area of RNA-mediated neurotoxicity, with the potential of leading to novel insights into neurodegeneration and to novel approaches to therapy. PUBLIC HEALTH RELEVANCE: The proposed experiments are designed to enhance our understanding of the pathogenesis of HDL2, and potentially of HD and DM1. More generally, the experiments will provide new understanding of the little explored area of RNA-mediated neurotoxicity, with the potential of leading to novel insights into neurodegeneration and to novel approaches to therapy.